A vesicle is also referred to as a liposome, and it is a small enclosed sac consisting of a lipid bimolecular membrane formed with amphiphilic lipid molecules and an internal water phase. Thus, it is anticipated that such a vesicle will be used for various purposes such as a model cell membrane, a DDS (drug delivery system) preparation, a food product capsule and a microreactor for research usage.
An example of the conventional method for producing a vesicle is a method comprising hydrating a thin lipid membrane formed on the surface of a solid. For example, a thin lipid membrane is formed on a glass substrate, and an aqueous solution that contains a substrate to be entrapped in a vesicle (hereinafter, referred to as an “entrapped substance”) is then added thereto, followed by intensive shaking, so as to produce a vesicle. However, the particle diameters of vesicles obtained by this method are generally non-uniform, and the entrapment efficiency of entrapping a desired entrapped substance in the vesicle (hereinafter referred to as “entrapment yield”) is extremely low. The term “entrapment yield” is used herein to mean “the ratio, expressed by percent by weight, of substances entrapped in a vesicle to those contained in the finally obtained vesicle suspension as a whole.” Moreover, it has also been difficult for this method to obtain a relatively large vesicle having a particle diameter of a micrometer-scale.
In order to enhance the entrapment yield of a desired substance in a vesicle, an emulsion can be used. For example, a method called “reverse phase evaporation method” disclosed in Non Patent Literature 1 has been known. In this method, a water phase containing a desired entrapped substance is dispersed in a volatile organic solvent containing an amphiphilic lipid, such as chloroform or ether, to produce a W/O emulsion, and only the solvent is removed from the produced emulsion by evaporation, and an aqueous solution is then added to the residue, so as to form a vesicle having a particle diameter of approximately 200 to 1,000 nm.
Moreover, as disclosed in Non Patent Literature 2, there has also been proposed a method for producing a vesicle having a particle diameter of approximately several hundreds of nm, which comprises: dispersing a water phase containing a desired entrapped substance in a volatile organic solvent containing an amphiphilic lipid, such as chloroform or ether, to produce a W/O emulsion; then producing a W/O/W emulsion by a stirring emulsification method using water or a buffer as an external water phase solution; and then removing the organic solvent from the produced W/O/W emulsion. According to such a method, there is a case in which a relatively high entrapment yield, such as an entrapment yield of 30% to 60%, may be achieved, and thus, this method is currently used as a method effective for entrapping a substance in a vesicle. Furthermore, Non Patent Literature 3 includes a report regarding the entrapment yield of a hydrophilic substance.
Patent Literature 1 by the present inventors discloses an effective method for producing a vesicle comprising: producing a W/O emulsion from a water phase containing an entrapped substance and an oil phase containing an emulsifier; freezing only an internal water phase; and then substituting the aforementioned emulsifier with a lipid, wherein this method differs from the method of the present invention.
Prior Art
Patented Documents
    Patent Literature 1: Japanese Patent No. 4009733Non-Patent Documents    Non Patent Literature 1: Szoka et al.: Proc. Natl. Acad. Sci. USA, vol. 75, No. 9, 4194-4198, 1978.    Non Patent Literature 2: Ishii et al.: J. Dispersion Sci. Technol., vol. 9, No. 1, 1-15, 1988.    Non Patent Literature 3: Nii and Ishii. Int. J. Pharmaceutics, vol. 298, 198-195, 2005